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SOX9 Is an Astrocyte-Specific Nuclear Marker in the Adult Brain Outside the Neurogenic Regions

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SOX9 Is an Astrocyte-Specific Nuclear Marker in the Adult Brain Outside the Neurogenic Regions The Journal of Neuroscience, April 26, 2017 • 37(17):4493–4507 • 4493 Cellular/Molecular SOX9 Is an Astrocyte-Specific Nuclear Marker in the Adult Brain Outside the Neurogenic Regions X Wei Sun,1 X Adam Cornwell,1 Jiashu Li,1 XSisi Peng,1 M. Joana Osorio,1 XNadia Aalling,2 Su Wang,1 Abdellatif Benraiss,1 Nanhong Lou,1 Steven A. Goldman,1,2 and XMaiken Nedergaard1,2 1Center for Translational Neuromedicine, University of Rochester Medical Center, Rochester, New York 14642, and 2Center for Basic and Translational Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen 2200 Copenhagen, Denmark Astrocytes have in recent years become the focus of intense experimental interest, yet markers for their definitive identification remain both scarce and imperfect. Astrocytes may be recognized as such by their expression of glial fibrillary acidic protein, glutamine synthe- tase, glutamate transporter 1 (GLT1), aquaporin-4, aldehyde dehydrogenase 1 family member L1, and other proteins. However, these proteins may all be regulated both developmentally and functionally, restricting their utility. To identify a nuclear marker pathogno- monic of astrocytic phenotype, we assessed differential RNA expression by FACS-purified adult astrocytes and, on that basis, evaluated the expression of the transcription factor SOX9 in both mouse and human brain. We found that SOX9 is almost exclusively expressed by astrocytes in the adult brain except for ependymal cells and in the neurogenic regions, where SOX9 is also expressed by neural progenitor cells. Transcriptome comparisons of SOX9ϩ cells with GLT1ϩ cells showed that the two populations of cells exhibit largely overlapping gene expression. Expression of SOX9 did not decrease during aging and was instead upregulated by reactive astrocytes in a number of settings, including a murine model of amyotrophic lateral sclerosis (SOD1G93A), middle cerebral artery occlusion, and multiple mini- strokes. We quantified the relative number of astrocytes using the isotropic fractionator technique in combination with SOX9 immuno- labeling. The analysis showed that SOX9ϩ astrocytes constitute ϳ10–20% of the total cell number in most CNS regions, a smaller fraction of total cell number than previously estimated in the normal adult brain. Key words: astrocyte marker; astrocytes; SOX9; transcriptome Significance Statement Astrocytes are traditionally identified immunohistochemically by antibodies that target cell-specific antigens in the cytosol or plasma membrane. We show here that SOX9 is an astrocyte-specific nuclear marker in all major areas of the CNS outside of the neurogenic regions. Based on SOX9 immunolabeling, we document that astrocytes constitute a smaller fraction of total cell number than previously estimated in the normal adult mouse brain. Introduction Gourine et al., 2010; Takata et al., 2011), buffering potassium (Kuf- Astrocytes serve multiple functions, including modulating neuronal fler and Nicholls, 1966), taking up synaptic glutamate (Bergles and activity (Nedergaard, 1994; Parpura et al., 1994; Kang et al., 1998; Jahr, 1997), maintaining endothelial tight junctions (Ballabh et al., 2004), and clearing brain metabolic waste products (Iliff et al., 2012). In fact, neurons have lost many basic cellular functions in the course Received Oct. 15, 2016; revised Jan. 25, 2017; accepted Feb. 19, 2017. Authorcontributions:W.S.,S.A.G.,andM.N.designedresearch;W.S.,J.L.,S.P.,N.A.,andN.L.performedresearch; of evolution and may depend on their symbiotic relationship with M.J.O., S.W., and A.B. contributed unpublished reagents/analytic tools; W.S. and A.C. analyzed data; W.S., A.C., astrocytes for their function and survival. It is therefore not surpris- S.A.G., and M.N. wrote the paper. ing that astrocytic dysfunction likely contributes to a host of neuro- This work was supported by the National Institutes of Health and Adelson Medical Research Foundation (Grants logical diseases, including Alzheimer’s disease, epilepsy, Parkinson’s R01NS075177andHD076892toM.N.)andtheAmericanHeartAssociation(PredoctoralFellowship12PRE12030048 disease, amyotrophic lateral sclerosis (ALS), and Huntington’s dis- to W.S.). The authors declare no competing financial interests. ease (Benraiss et al., 2016; Verkhratsky et al., 2016), among others. W.Sun’spresentaddress:LaboratoryofMolecularandCellularNeurobiology,NationalInstituteofMentalHealth, However, the cellular composition of the central nervous system Bethesda, MD 20814. (CNS) is complex and identification of astrocytes, which are both Correspondence should be addressed to either Wei Sun or Maiken Nedergaard, Center for Translational Neuro- morphologically and functionally heterogeneous, has been challeng- medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Box 645, Rochester, NY 14642. E-mail: [email protected] or [email protected]. ing. Currently, a few astrocyte markers are used routinely, including DOI:10.1523/JNEUROSCI.3199-16.2017 glial fibrillary acidic protein (GFAP), S100 calcium-binding protein Copyright © 2017 the authors 0270-6474/17/374493-15$15.00/0 B (S100␤), glutamate transporter 1 (GLT1) (Rothstein et al., 1994), 4494 • J. Neurosci., April 26, 2017 • 37(17):4493–4507 Sun et al. • Astrocyte-Specific Nuclear Marker aquaporin-4 (AQP4), connexin-43 (Cx43), connexin-30 (Cx30) perfused with cold HBSS, and decapitated. The brain was immediately re- (Nagy et al., 1999), and, more recently, aldehyde dehydrogenase 1 moved and cortex was dissected and cut into small pieces, which were di- 2ϩ 2ϩ family member L1 (ALDH1L1) (Cahoy et al., 2008). However, even gested with 5 U/ml papain in Ca /Mg -free PIPES/cysteine buffer, pH 7.4, for1hat37°C/5% CO2. After one wash, the tissue was then further though transcription factors play key roles in cellular differentiation 2ϩ and drive expression of phenotype-specific genetic programs, the digested with 40 U/ml DNase I in Mg -containing MEM with 1% bovine serum albumin (BSA) for 15 min at 37°C/5% CO . The tissue was then expression of astrocytic transcription factors beyond early develop- 2 carefully triturated in cold MEM with 1% BSA and centrifuged over a 90% ment has not been studied extensively. Percoll gradient. Cells below and included in the lipid layer were collected, Several recent studies have suggested that the transcription factor washed, and centrifuged to collect the cell pellet. For wild-type mice, the cells SOX9 is highly enriched in astrocytes (Pompolo and Harley, 2001; were labeled with rabbit anti-GLT1 antibody (1:150, custom made; Invitro- Lovatt et al., 2007; Barnabe´-Heider et al., 2010; Molofsky et al., 2013; gen) or rabbit IgG control followed by labeling with secondary donkey anti- Zhang et al., 2014;Farmer et al., 2016;Nagao et al., 2016;Zhang et al., rabbit allophycocyanin (APC) (1:200). Cells were resuspended in cold MEM 2016). On that basis, here, we examined the expression pattern of with 1% BSA containing 5 ␮g/ml 4Ј,6-diamidino-2-phenylindole (DAPI) to SOX9 in both young and aged wild-type mice, as well as in several discriminate dead cells. models of reactive astrogliosis, using genomics, immunohistochem- Cells were sorted using BD FACSAria III Cell Sorting System immedi- istry, and transgenic reporter mice. Comparisons of the transcrip- ately. Single cells were discriminated using pulse width and height mea- tomes of SOX9ϩ cells and GLT1ϩ cells indicated that these two surements. APC was excited by a 633 nm laser, and emissions were collected by a 660/20 nm band-pass filter. EGFP was excited by a 488 laser populations exhibited largely overlapping gene expression. Like and emissions were collected by a 530/30 nm band-pass filter and DAPI other currently used astrocytic markers, SOX9 specifically labels as- was excited by a 407 nm laser and emissions were collected by a 450/40 trocytes outside of the neurogenic regions: the subventricular zone band-pass filter. Dead cells were excluded according to positive DAPI (SVZ), the subgranular zone (SGZ) of the hippocampal dentate signal. GLT1ϩ and GLT1Ϫ populations were gated according to isotype gyrus, and the rostral migratory stream (RMS) of the olfactory bulb. control. Cells were sorted into cold MEM containing 1% BSA. However, SOX9 also manifested several advantages in that respect, RNA processing, microarray, and quantitative PCR (qPCR). After including that its expression was not diminished by age or functional FACS, cells were immediately extracted for total RNA using either the status. It did not decrease during aging, remained nuclear in local- RNAqueous Micro kit (Life Technologies) or RNeasy Micro Kit (Qia- ization in mature astroglia, and was upregulated in scar tissue, all of gen). Mouse cortical and spinal cord tissue samples were collected after which facilitated the identification of individual astrocytes. Indeed, transcardial perfusion of ice-cold HBSS and RNA was extracted with RNeasy Plus Universal Mini Kit (Qiagen). RNA quantity was assessed quantification of the relative number of astrocytes, based on com- using the NanoDrop 1000 and RNA integrity was assessed using an Agi- bining the isotropic fractionator technique with SOX9 immunola- lent Bioanalyzer 2100. For microarray, 20 ng of total RNA was amplified ϩ beling (Herculano-Houzel and Lent, 2005), showed that SOX9 and labeled with biotin using the Ovation kit according to the manufac- astrocytes constitute only ϳ10–20% of total cells in most CNS re- turer’s instructions and hybridized to the Affymetrix GeneChip Mouse gions and
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